Adjustments to cardiac output are made on a beat-to-beat basis in response to internal and external stimuli, and are mediated by the changing balance of input from the sympathetic and parasympathetic branches of the autonomic nervous system. While parasympathetic input normally tempers the pro-arrhythmic influence of sympathetic activation, too much parasympathetic influence can predispose to atrio-ventricular block or atrial fibrillation (a key risk factor for stroke). Conversely, too little parasympathetic influence is associated with heart failure, and is an independent predictor of increased morbidity and mortality in patients with coronary artery or congenital heart disease, and following myocardial infarction. Collectively, these observations suggest that altered parasympathetic regulation of the heart likely contributes to a broad spectrum of cardiac dysfunction, while simultaneously highlighting the inherent therapeutic promise of manipulations designed to predictably alter parasympathetic tone in the heart. The premise of this project is that the diagnostic and therapeutic potential associated with the parasympathetic regulation of the heart cannot be fully-realized without a clearer understanding of the signaling pathway(s) mediating its influence. A critical signaling pathway mediating the parasympathetic influence on the heart consists of the type 2 muscarinic acetylcholine receptor (M2R) and the G protein-gated potassium channel IKACh; the M2R-IKACh signaling pathway is a major determinant of cardiac rhythmicity, and dysregulation of this pathway has been implicated in both atrial and ventricular arrhythmias. Work in the initial project period established that the RGS6/G5 complex accounts for much of the RGS- dependent negative modulation of the M2R-IKACh signaling pathway, and that loss of this inhibitory influence yields enhanced parasympathetic signaling in the heart. The goals for the next project period are articulated in two Specific AIMs that are designed to: (1) Identify molecules and mechanisms underlying the RGS modulation of parasympathetic signaling, and (2) Develop targeted approaches to manipulate parasympathetic signaling in the heart. The proposed studies employ innovative and complementary approaches that leverage the unique strengths and reagents of two research labs, along with the expertise of an extended group of collaborators with overlapping research interests. Successful completion of the proposed work will yield a detailed understanding of the RGS-dependent modulation of parasympathetic signaling in the heart, along with tangible insights into the therapeutic potential associated with direct pharmacologic manipulation of the IKACh channel. Collectively, these efforts will result in a sophisticated understanding of the molecular and cellular mechanisms that shape the parasympathetic regulation of the heart, knowledge that can be translated into improved diagnostic and therapeutic approaches for arrhythmias.